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Juxtaposition of the genes encoding Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW3
1Department of Microbiology, University of Georgia, Athens 30602.
Abstract:
The loss and reacquisition of high-Mr (HMW) proteins, HMW1, 2, 3, 4 and 5, by Mycoplasma pneumoniae correlates with cytadherence phase variation. We are cloning and characterizing the genes encoding HMW1-5 to understand the mechanism regulating their coordinate expression. HMW1 was purified by polyacrylamide-gel electrophoresis. Amino acid (aa) sequence data were obtained from enzymatically generated peptide fragments from HMW1. A degenerate 17-mer probe synthesized based upon the aa sequence of one peptide clearly identified a single 4.75-kb BamHI fragment of M. pneumoniae DNA under stringent hybridization conditions. This fragment was cloned into pUC19 to generate pKV16. Restriction mapping of the 4.75-kb BamHI fragment in pKV16 revealed a possible overlap with the 9.4-kb EcoRI fragment containing the gene encoding protein HMW3. Southern blotting and reciprocal hybridization studies confirmed this overlap, establishing the juxtaposition of the genes encoding HMW1 and HMW3. Finally, physical mapping analysis by probing restriction fragments of M. pneumoniae DNA resolved by pulsed-field gel electrophoresis with the cloned genes encoding HMW1 and HMW3 revealed definitively that the hmw locus maps to a 106.8-kb ApaI fragment, rather than a 117.5-kb ApaI fragment, as had been reported previously for hmw3 [Krause and Mawn, J. Bacteriol. 172 (1990) 4790-4797].
Insights
Mycoplasma pneumoniae phase variation involves high-molecular-weight (HMW) proteins. This study clones and maps genes for HMW1 and HMW3, revealing their adjacent location within the hmw locus.
Area of Science:
- Molecular microbiology
- Bacterial genetics
- Protein biochemistry
Background:
- Mycoplasma pneumoniae exhibits phase variation in cytadherence, linked to the expression of high-molecular-weight (HMW) proteins (HMW1-5).
- Understanding the genetic basis of HMW protein expression is crucial for deciphering cytadherence regulation.
Purpose of the Study:
- To clone and characterize genes encoding HMW1-5 proteins.
- To elucidate the mechanism regulating the coordinate expression of these HMW proteins.
- To determine the precise genetic locus of HMW1 and HMW3 genes.
Main Methods:
- Purification of HMW1 protein using polyacrylamide-gel electrophoresis.
- Amino acid sequencing of peptide fragments from HMW1 to design degenerate DNA probes.
- Southern blotting, DNA fragment cloning (pUC19), restriction mapping, and pulsed-field gel electrophoresis for gene mapping.
Main Results:
- A 4.75-kb BamHI DNA fragment containing the HMW1 gene was identified and cloned.
- The HMW1 and HMW3 genes were found to be juxtaposed, sharing a common genomic region.
- Physical mapping confirmed the hmw locus resides on a 106.8-kb ApaI fragment, correcting previous reports.
Conclusions:
- The genes encoding HMW1 and HMW3 are located adjacent to each other.
- This study refines the physical map of the M. pneumoniae genome concerning the hmw locus.
- Provides foundational data for understanding the coordinated regulation of HMW proteins in M. pneumoniae.